Carotenoids are deep yellow-orange pigments found in orange and yellow colored vegetables and in many dark green foods. Beta carotene is the most abundant of the various carotenoids. Beta carotene can be converted by the body to vitamin A. Vitamin A is a fat soluble vitamin that can be stored in the body for a limited period of time, primarily in the liver, unlike the water soluble vitamins, which are not storable. Vitamin A can be toxic if ingested in large amounts. However, beta carotene is converted by the body into vitamin A as needed and typically is considered a non-toxic source of vitamin A even in large amounts.
Beta carotene has been identified as an antioxidant that is capable of countering the damaging effects of oxidation in animal tissues. For this reason and as a nontoxic source of vitamin A, beta carotene has been highly valued and commercially important as a nutritional supplement. However, concerns have recently arisen regarding the health benefits of beta carotene as a nutritional supplement isolated from the mixed carotenoids in which beta carotene typically is found in nature.
Plant derived mixed carotenoids, including beta carotene, can be obtained from a number of sources including carrots, spinach, and palm oil, but their relative concentration is higher in algae of the genus Dunaliella. These algae are commonly found in concentrated salt solutions. Under appropriate growth conditions, more than ten percent of the algal dry weight can be mixed carotenoids.
For example, Dunaliella salina tends to accumulate significant amounts of carotenoids and glycerol when stressed by exposure to high temperatures, intense light, and brine solutions having a concentration of sodium chloride of greater than about 20 percent by weight per unit volume of brine. The carotenoids are thought to protect the algae from sunlight. The concentration of carotenoids increases with increasing salt concentration of the brine up to the limit of halotolerance of the algae.
Numerous methods have been proposed for recovering beta carotene, carotenoids, and other valuable components from Dunaliella salina. Dunaliella salina provides a source of beta carotene and other carotenoids from which several nutritional supplements presently are manufactured. However, economically efficient recovery of carotenoids from Dunaliella salina in a form free from potentially toxic solvents and other undesirable substances has been somewhat problematic. Commercial exploitation of Dunaliella salina as a source of carotenoids presents numerous difficulties.
Halotolerant algae, including Dunaliella salina are typically found in salt lakes, including the Great Salt Lake in Utah. Harvesting Dunaliella salina from lakes and other natural settings typically is not commercially practicable, in part because of the low concentrations that are found in uncontrolled growing conditions.
Commercially, Dunaliella salina normally is harvested from cultures that are produced in specially constructed outdoor ponds. The outdoor ponds typically are constructed in regions with a hot and arid climate with little rainfall and few cloudy days to promote carotenoid production.
Two distinct methods of aquaculture have been developed for growing algae. These are an intensive mode and an extensive mode. Both aquacultural techniques require the addition of fertilizers to the medium to supply the necessary inorganic nutrients, phosphorous, nitrogen, iron, and trace metals, that are necessary for biomass production through photosynthesis.
The primary difference between the two modes of production is mixing of the growth medium. Intensive ponds employ mechanical mixing devices while extensive ponds rely on mixing by the wind. Therefore, factors that affect algae growth can be more accurately controlled in intensive aquaculture.
In both the intensive and extensive modes, the salinity of the growth medium is controlled within a specified range, usually between about 18 to 27 percent sodium chloride by weight per unit volume of brine. This range of concentrations is thought to provide the maximum carotenoid production. The optimum growth range for Dunaliella salina is said to be between about 18 and 21 percent salinity. Maximum carotenoid production in the algal biomass is said to occur at salinities greater than about 27 percent. Maximum carotenoid production per unit volume of the brine medium has been reported to occur at about 24 percent salinity.
Outdoor ponds for intensive aquaculture typically are somewhat expensive and are frequently constructed of concrete and lined with plastic. Brine depth generally is controlled at 20 centimeters, which has been considered to be the optimum depth for producing algal biomass. A number of configurations of the ponds have been proposed for intensive aquaculture. However, the open air raceway ponds are typically the most important commercially. Raceway ponds employ paddle wheels to provide mixing. Chemical and biological parameters are carefully controlled, including salt and fertilizer concentrations, pH of the brine, and purity of the culture.
Extensive aquaculture has been practiced in the hot and arid regions of Australia. Outdoor ponds for extensive aquaculture generally are larger than those for intensive aquaculture and normally are constructed in lake beds. The open air ponds are typically bounded by earthen dikes. No mixing devices are employed. Mixing in the pond is generated by the wind. Pond depth and chemical composition are optimized for maximum carotenoid production.
However, the parameters for maximum carotenoid production and culture purity and stability are not as easily controlled in the extensive ponds as in the intensive ponds because of the lack of efficient mixing and the larger volume of the extensive pond. The composition of the brine fluctuates. The algal biomass is less concentrated than in the intensive ponds. The extensive pond is more susceptible to infestations by predators and competitors.
Predators and competitors cannot typically survive at salinities of about 20 percent and above. If the salinity of the pond drops below about 20 percent, the culture may become infested with a predator that may rapidly increase in number and decimate the Dunaliella salina population. The primary predators are the ciliated protozoan Fabrea salina and the brine shrimp Artemia salina. At salt concentrations below about 15 percent, other algae tend to compete with Dunaliella salina for nutrients and additional predators may further reduce the Dunaliella salina population.
Recovery of the algae from the brine is more problematic in the extensive pond than in the intensive pond because of the more dilute culture. However, it has been observed that algae tend to concentrate in windrows at the edges of extensive ponds and in natural salt lakes. The algae are often blown across the surface of the lake or pond where they collect and concentrate in windrows at the lee side. It has been recognized that the ability to harvest the windrows could significantly improve the process economics because of the higher concentration of algae. Nevertheless, satisfactory techniques for harvesting windrows are not generally available.
It is not usually possible to consistently harvest windrows from a fixed harvesting plant site. Wind direction normally is somewhat unpredictable and may change frequently. The windrows may form at different locations along the side of the pond or lake. When the windrow does not form at a fixed harvesting plant site, then a dilute suspension that is depleted in the alga is processed, which results in a reduced production rate. Harvesting costs are higher due to the processing costs associated with more dilute cultures.
Nevertheless, higher harvesting costs may be offset by the capital costs associated with constructing concrete and plastic lined ponds for intensive aquaculture. Pond construction costs per unit volume for the earthen extensive ponds are significantly lower than those for the lined concrete ponds of intensive aquaculture.
It has been recognized that if algae could be harvested from the lakes in which they grow naturally, then pond construction costs, fertilizer costs, and brine make up costs conceivably could be substantially eliminated. However, harvesting algae from lakes and other natural settings typically has been considered uneconomical and without commercial utility. There usually is no degree of control over the salinity of the lake waters, the mineral and nutrient composition of the lake waters, and the degree of mixing in a natural salt lake. Dilute cultures of algae of questionable stability may occur.
Dilute cultures of Dunaliella salina are generally uneconomical to process in part because of the problems and difficulties encountered in separating the algae from the brine in which they grow. The algae have mobility, neutral density, and a small elliptical shape of approximately 12 to 16 microns by 25 microns that makes the algae somewhat difficult to harvest.
Dunaliella salina typically is separated from the brine within which it is found by using a chemical flocculating or coagulating agent in combination with a settler, centrifuge, filter, adsorbent, or other separation means. Chemical treatments, including, for example, the silanes, can be applied to adsorption media to enhance adsorption. Various processes have been proposed for extracting beta carotene, carotenoids, and other valuable components from the algae, including glycerol and proteins. Hydrocarbon solvents, edible oil solvents, and supercritical carbon dioxide have been proposed as solvents for the extractions. The algae may be disintegrated by mechanical means to promote extraction of the components.
Chemical additives such as flocculants and coagulants have limited the commercial exploitation of Dunaliella salina as a source of carotenoids and beta carotene in part because of the costs of adding these components to algal suspensions, particularly dilute suspensions. Chemical additives, chemical treatments, and hydrocarbon solvents are considered undesirable in nutritional supplements.
It would be desirable to more economically and efficiently harvest Dunaliella salina and to extract the carotenoids and other valuable components therefrom with minimal or no undesirable additives.